Our basic research has been focused on RNA-processing proteins [RNase III (model system for a family of dsRNA-specific endonucleases exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer) and Era (conserved GTPase that couples cell growth with cell division)] and RNA polymerase (RNAP)-associated transcription factors [RapA (ATP-dependent dsDNA translocase that recycles RNAP during transcription) and N-utilizing substances A, B, E, and G (NusA, NusB, NusE, and NusG)]. Previously, we made pioneering contribution to the mechanism of RNase III action and a breakthrough advance in the structure and functional cycle of Era. We also determined the crystal structure of RapA and a plectonemic RNA supercoil, and provided structural insights into the phage lambda N protein-mediated transcription antitermination by determining crystal structures of the ternary NusB-NusE-BoxA RNA and NusB-NusE-dsRNA complexes. This year, our most significant accomplishment is the crystal structure of the Saccharomyces cerevisiae RNase III (Rnt1p) post-cleavage complex that explains why Rnt1p binds to RNA stems capped with an NGNN tetraloop. The structure shows specific interactions between a structural motif located at the end of Rnt1p dsRNA-binding domain (dsRBD) and the guanine nucleotide in the second position of the loop. Strikingly, structural and biochemical analyses indicate that the dsRBD and N-terminal domains (NTD) of Rnt1p function as two rulers that measure the distance between the tetraloop and the cleavage site. These findings provide a framework for understanding eukaryotic RNase IIIs, including Drosha and Dicer. This study is published as a featured research article in Molecular Cell and highlighted on the issue cover of the journal. Our effort in structure-based drug development has been focused on Glutathione S-transferase (GST)-activated, nitric oxide-releasing anticancer prodrugs and bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase (HPPK) useful as antibacterial agents. Previously, our structure-based design of prodrugs yielded PABA/NO, which exhibits anticancer activity both in vitro and in vivo with potency similar to that of cisplatin. We also designed, synthesized, and characterized a group of HPPK inhibitors as lead compounds for novel antibiotics, and optimized the synthetic route of HPPK inhibitors, leading to the invention of a novel intermediate and a new method for the synthesis of a known intermediate with a yield of 95%. This year, we have elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against Francisella tularensis (Ft) HPPK that is combined with dihydropteroate synthase (DHPS), determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/260,000 that of Escherichia coli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing tularemia therapeutics and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of 10 F. tularensis genomes indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because mutation may make the marginal DHPS activity unable to support the growth of F. tularensis. This work is published in the FEBS Journal, the special issue celebrating the International Year of Crystallography 2014.